作者は感謝して、米国森林局森林健康保護の評価の監視と発展を支えてきた特殊な技術開発プログラム、強く、ブナカイガラムシの影響を受けやすい木を識別するために、ここで説明されたプロトコルの変更やアプリケーションからの資金調達を認める。また、当社のアメリカブナ抵抗スクリーニングプログラムの彼らの継続的なパートナーシップとサポートのため、オハイオ州カートランド、ホールデン樹木園に感謝しています。

1植物材料：熟フィールド木、鉢植え苗、または鉢植え移植片<ol><li>フィールドテストでは、可能な抵抗のためのテストのためのスケールの侵入や病気の兆候を示さない、成熟した健康的なアメリカのブナの木を選択します。目に見えて影響を受けやすい木はまた、対照（ 図1）として使用されるように識別される必要があろう。 </li><li>鉢植えの苗や移植片をテストするための、収集·コッホ＆キャリー、2004またはキャリーら 、2013に記載されたグラフト穂に記載されているようにbeechnuts発芽。 </li><li> 47グラムの微量栄養素​​、477グラム徐放性肥料15N-3.9P-9.9K、700グラムの粗パーライトと2.8立方あたり75グラムの硫酸アルミニウムを改正ポッティング土壌ミックスの苗や移植片を育てる。フィートバッグ。生育期の後半で必要に応じて、200 ppmの窒素において、可溶性17N-1.3P-14.1K毎週植物を肥やす。 </li><li>生育期間中は、日陰​​の家の中で植物を保つ。植物は前にそれらを移動する秋に休眠外に行くことを許可する制御された温度貯蔵施設（〜4℃）に11月から4月を通して。 </li></ol>ブナスケール卵の2。コレクション<ol><li> BBDが出没するスタンドでは、八月中旬まで月中旬から通常豊富な卵の存在を確認するために手虫眼鏡で（簡単に「白い」外観、 図1Aで識別される）に大きくはびこっ木を検査します。 </li><li>プラスチック密封可能な1ガロンの収集袋（ 図1B）に、ゆっくりと大人のカイガラムシ、卵やその他の破片の白いワックス状の塊を磨くために絵筆を使用しています。少なくとも12メートル離れて三つの異なる木の最小値から収集します。 </li><li>必要に応じて、4℃で2週間まで密封された収集袋に卵を保管乾燥から卵を防ぐために、バッグの内側に湿らせたポリエチレンフォームのテープの小片（2.5cm角）。 </li><li>大人（0.60ミリメートル）とdebriからスケールの卵（0.15×0.25ミリメートル）を分離するS、2 &#39;塩ビ管の短い部分と250ミクロンのナイロンメッシュの正方形の一枚（ 図2）をサポートするカップリングを使用してふるいを構築する。 </li><li>ふるい上に収集袋から大人、ワックス、卵、破片の混合物を空にして静かに以下のガラスシャーレにメッシュを通過するように卵を奨励するために、小さなペイントブラシを使用しています。彼らはそれが困難に卵を移動すること、より多くの静電気を保持するため、プラスチック製ペトリ皿は避けるべきである。卵の前と後の篩い分けを図3に示す。 </li><li>精製された卵は、少なくとも1週間、4℃でペトリ皿に格納することができる。乾燥から卵を防ぐために、蓋に泡の湿った部分をテープで固定し、パラフィルムで密封する。 </li></ol> 3。卵生存率アッセイ<ol><li>卵の生存率を評価するために、ガラスを60mmペトリ皿（ 図4Aの底部の周囲にワセリンの薄いリングを適用するために10ccのシリンジを使用し</strongの&gt;）。 </li><li> 、リングの中心に、約100の卵を移すシャーレにふたを置いて、パラフィルムで密封する。卵は事前のアッセイ開始まで4℃で保存した場合に密封されたペトリ皿で​​2週間、3週間、室温で邪魔されずに残ることができます。 </li><li>孵化し幼虫は、石油ゼリー中のスタックになり、簡単にカウントすることができ、空の卵は簡単に自分の色や光沢（ 図4B）で孵化していない卵から区別されます。空の卵を加え、残りのフル卵の合計によって幼虫の数を割ることによって実行可能性を計算する。良好な生存率は75％〜90％の孵化した卵の範囲であるべきである。 </li></ol>分野における大規模な熟ぶなの4。スケール抵抗上映<ol><li>定量試験のため、解剖顕微鏡を使用して、小さなへらを使って500の卵を数える優しくポリエチレンフォームのpredampened、オープンセル10×15×1.3センチメートルの長方形の中心を横断して振りかける。へ泡を減衰させる、できるだけ多くの水を絞り出す、それを濡らす。定性試験では、500卵カウントアウトすることができ、小さなガラスバイアルに、約500卵の追加のバッチを測定するために使用することができる描か「充填」の行に配置された。 </li><li>樹皮に対向する卵の側にテストツリー上にフォームパッドを配置します。ロープ、ひも、より糸、プラスチックコーティングされたハードワイヤーで所定の位置にパッドを保持する。プラスチックや金属系材料ではなく、より容易に野生動物により除去された天然繊維材料よりも使用する必要があります。 </li><li>アセテート系シリコーン接着剤に沿って蒸気透過性防水ハウスラップの上部とプレカットの側面プリント23 x 30 cm片と泡試験パッド上に置きます。防水シールを作成するために木にハウスラップの端を押してください。しばらく接着剤セット（ 図5）所定の位置に保持するために、木とハウスラップの周りにナイロンひもやプラスチックコーティングされたハードワイヤーを配置します。 </li><li> 2の最小値を置き好ましくは木の幹の両側の各テストツリー上のテスト·パッド、。各サイトで、対照として（明白な自然のスケールの侵入で）少なくとも2の影響を受けやすい木にテストパッドを配置。対照木にテストパッドを配置する前に、会社を使用して、任意の天然に存在するカイガラムシや卵を取り除くには、ブラシを毛先。 注：卵フォームパッドが蔓延レベルが低い地域では特に役立ちますスケールの卵を、飼育する目的のために木にも配置することができます。 </li></ol>鉢植えの苗や移植片の5。スケール抵抗上映<ol><li>テストのために、それらの上に配置された少なくとも2つの別々のテストパッドを持っているのに十分な高、1センチメートル（5センチメートル土壌線の上キャリパー）の最小径と鉢植えの木を選択します。必要なときにテストパッド用のスペースを確保するために、小さな側枝を剪定。 </li><li> 150卵を数え、プレカットの上にそれらを散乱させる解剖顕微鏡を使用し、2.5×7.6 X 1.3センチメートル気泡ポリエチを湿らレンのフォームパッド。 </li><li>プラスチック被覆ワイヤーを使用して、樹皮に対する卵の側に苗ためにフォームパッドを貼付。ちょうど泡の上に苗の周りのハウスラップの小さな正方形をラップした（ 図6Aを参照）アセテート系シリコーンでそれを封印。 </li><li>コントロールとして苗家族や移植片の影響を受けやすい知ら含まれています。 </li></ol> 6。データ収集注：約52から57週の試験木にフォームパッドと卵を置いた後、データを収集することができる。なお、これは大人が再生する能力を決定することができるように、卵を産み始めた後まで行われないことが重要である。いくつかの耐性の木にそれは成人の数が少ないが、全く再生が発生していないとの確立を参照することも珍しくありません。 <ol><li>慎重にフォームパッドを取り外し、手レンズや拡大鏡（10倍）を使用して、樹皮上に確立大人のカイガラムシの数を数える。 </li><li>フォームパッドが除去されると、それはuのではないいくつかの大人と泡と木の外に引っ張られる最も卵のクラスタ用ncommonます（ 図7を参照）。解剖顕微鏡を使用して、フォームパッドに付着し残っている卵のクラスタと大人を数える。 </li></ol>

<ol>
	<li>Houston, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+artificially+infest+beech+bark+with+the+scale,+Cryptococcus+fagisuga+(Lindinger).">A technique to artificially infest beech bark with the scale, Cryptococcus fagisuga (Lindinger).</a> U.S. Forest Service. , (1982).</li><li>Houston, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+beech+resistance+to+Cryptococcus+fagisuga.">American beech resistance to Cryptococcus fagisuga.</a> Proceeding, IUFRO beech bark disease working party conference. , 38-42 (1983).</li><li>Ehrlich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beech+bark+disease:+a+nectria+disease+of+Fagus,+following+Cryptococcus+fagi.">The beech bark disease: a nectria disease of Fagus, following Cryptococcus fagi.</a> 10, 593-692 (1934).</li><li>Papaik, M. J., Canham, C. D., Latty, E. F., Woods, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+an+introduced+pathogen+on+resistance+to+natural+disturbance:+beech+bark+disease+and+withdrow.">Effects of an introduced pathogen on resistance to natural disturbance: beech bark disease and withdrow.</a> 35, 1832-1843 (2005).</li><li>Miller-Weeks, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+beech+bark+disease+in+New+England+and+New+York.">Current status of beech bark disease in New England and New York.</a> Proceeding, IUFRO beech bark disease working party conference. , 21-23 (1983).</li><li>Houston, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beech+bark+disease:+The+aftermath+forests+are+structured+for+a+new+outbreak.">Beech bark disease: The aftermath forests are structured for a new outbreak.</a> J. For. 73, 660-663 (1975).</li><li>Twery, M. J., Patterson, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variations+in+beech+bark+disease+and+its+effects+on+species+composition+and+structure+of+northern+hardwood+stands+in+central+New.">Variations in beech bark disease and its effects on species composition and structure of northern hardwood stands in central New.</a> 14, 565-574 (1984).</li><li>Jakubus, W. J., McLaughlin, C. R., Jensen, P. G., McNulty, S. A., Evans, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternate+year+beechnut+production+and+its+influence+on+bear+and+marten+populations.">Alternate year beechnut production and its influence on bear and marten populations.</a> Beech Bark Disease: Proceedings of the Beech Bark Disease Symposium. , 79-87 (2005).</li><li>Koch, J. L., Carey, D. W., Mason, M. E., Nelson, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+beech+scale+resistance+in+full-+and+half-sib+families.">Assessment of beech scale resistance in full- and half-sib families.</a> Can. J. For. Res. 40, 265-272 (2010).</li><li>Koch, J. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fifty+year+impacts+of+the+beech+bark+disease+in+the+Bartlett+Experimental+Forest,+New+Hampshire.">Fifty year impacts of the beech bark disease in the Bartlett Experimental Forest, New Hampshire.</a> Northern J. Appl. For. 23, 141-143 (2006).</li><li>Koch, J. L., Carey, D. W., Yaussy, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cross-pollinations+with+American+beech+trees+that+are+resistant+to+beech+bark+disease.">Controlled cross-pollinations with American beech trees that are resistant to beech bark disease.</a> Proceedings of the 14th Central Hardwood Forest Conference. , 358-364 (2004).</li><li>Carey, D. W., Mason, M. E., Bloese, P., Koch, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+callusing+for+propagation+of+American+beech+by+grafting.">Hot callusing for propagation of American beech by grafting.</a> HortSci. 48, 620-624 (2013).</li><li>Koch, J. L., Carey, W. W., Evans, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+resistance+of+American+beech+to+beech+bark+disease:+knowledge+through+2004.">The genetics of resistance of American beech to beech bark disease: knowledge through 2004.</a> Beech Bark Disease: Proceedings of the Beech Bark Disease Symposium. , 98-105 (2005).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

このアッセイの成功のために必要な重要なステップは、卵と試験された各サイトで、鉢植えの材料との影響を受けやすいコントロールの使用上の実行可能性テストを実行することを含む。これは、木または実生ごとに複数のテスト用パッドを使用することも重要である。我々は、フィールド内の鉢植えの植物に、より制御された試験の両方において、頻繁なエラーの源は、パッド不良である?...

ブナ樹皮病（BBD）が1890年代後半3ノバスコシア州のカナダの州では、侵襲的なブナカイガラムシの導入以来、北米でクリプトコッカスfagisugaをアメリカのブナに有害な影響を与えました。ブナカイガラムシが真菌（NeonectriaのditissimaまたはNeonectriaのfaginata）のNeonectria種の 1による感染のために玄関を提供する小さな亀裂を作成樹皮にその送りスタイを挿入すると、この昆虫病錯体が開始されます。真菌菌糸が成長するにつれ、組織の大部分は、最終的には完全に木を環状剥皮、死ぬかもしれない。病気による被害は強風4でスナップにそれが発生しやすいこと、木を弱める。疾患の最初の波の死亡率レベルは50％5と高いことが報告されている。潰瘍は、木材製品としてツリーの値を小さく形成するように、生存している木はしばしばひどく変形される。0;このような木は、確立スタンド6の経済的、生態学的価値を減らすことから、他のより望ましい種を防ぐ「ブナの藪」の形成につながるルート発芽する傾向を持っている。ブナ樹皮病はアメリカのブナの絶滅につながる可能性はありませんが、それは野生生物の7,8のために食料や生息地の減少につながる物と健康をスタンド変える。 何年も前からBBDの影響を受け、スタンドでは、疾患の症状がないままの木が​​報告されている。人工接種試験は、これらの木がカイガラムシ2に耐性であることが示されている。スケールの侵入がないと、Neonectria感染の機会がほとんどないが、真菌の影響を最小限に抑えることがある。前のスケールの侵入がない場合のNeonectriaの感染によるアメリカのブナにおける大規模な死亡率は決してありません報告されたので、BBDへの抵抗におけるブナのスケール昆虫結果に対する抵抗されて。 BBDの管理に関する最近の研究では、識別、繁殖、飼育、およびブナカイガラムシに対する抵抗性とアメリカのブナの木の保持に焦点を当てている。遺伝学的研究は、カイガラムシに対する耐性が遺伝性であり、耐木の慎重な選択および繁殖は、単一世代9の有意な改善をもたらすことができることを示している。この知見は、復元作付け10,11のための遺伝的に多様なBBD耐性種子の供給源を提供するために、耐性のアメリカのブナの地域採種園を確立するために、米国の州および国家森林管理者の努力に燃料を供給しています。研究はまた影響を受けやすい樹木や耐性樹木の維持を除去することにより、スタンドの遺伝学の育林操作はスタンド改良9,12をもたらすことができることを示した。 &lt;/ pの&gt; 木の改善活動を通して、または造林処方を行うを通じてBBDの管理はのために選択し、ブナのスケール性と感受性の木々の間を区別する能力を必要とします。ここで紹介する方法は、まず、人工的にブナのスケールの卵1を苗に接種するためにデイブヒューストンによって導入方法から適応されている。この方法は、耐性に関連する量的形質遺伝子座（QTL）を同定するために、または遺伝的研究に耐性と感受性の鉢植え苗木又はグラフト化ラミートを区別するためのスクリーニングツールとして使用することができる。あるいは、採種園の開発、またはフィールドでの保持のために耐性ツリーを識別するためにフィールド内の成木をスクリーニングするために使用することができる。感受性の樹木を同定し、疾患の影響を最小限にするために除去することができる。

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			<td height="63" rowspan="2" style="height:63px;width:163px;">Nylon Mesh Sheet, 250 micron</td>
			<td rowspan="2" style="width:120px;">Small Parts</td>
			<td rowspan="2" style="width:99px;">CMN-0250-D</td>
			<td style="width:627px;">Mesh opening size: 250 microns square, % of open area: 34, thread diameter: 180 micron, width: 24&#8221;, Length: 12&#8221;, Package quantity: 1</td>
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			<td height="21" style="height:21px;width:627px;"><a href="http://www.amazonsupply.com/dp/B000FMWH1S/ref=sp_dp_g2c_asin">http://www.amazonsupply.com/dp/B000FMWH1S/ref=sp_dp_g2c_asin</a></td>
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			<td height="42" rowspan="2" style="height:42px;width:163px;">Tyvek Home Wrap</td>
			<td rowspan="2" style="width:120px;">DuPont</td>
			<td rowspan="2" style="width:99px;">D1349991</td>
			<td style="width:627px;">9 ft x 150 ft roll, can ask for it to be cut in half.</td>
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			<td height="21" style="height:21px;width:627px;"><a href="http://www.lowes.com/pd_14582-2020-D13499991_0__">http://www.lowes.com/pd_14582-2020-D13499991_0__</a></td>
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			<td height="42" rowspan="2" style="height:42px;width:163px;">Polyethylene foam, &#189;&#8221; thick&#160;</td>
			<td rowspan="2" style="width:120px;">Columbus Foam Products</td>
			<td rowspan="2" style="width:99px;">N/A</td>
			<td style="width:627px;">2 ft x 50 ft roll</td>
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			<td height="21" style="height:21px;width:627px;"><a href="http://www.colsfoam.com/apcfp/products.htm">http://www.colsfoam.com/apcfp/products.htm</a></td>
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			<td height="64" rowspan="2" style="height:64px;width:163px;">MetroMix 510</td>
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			<td style="width:627px;">2.8 cu. ft. bark, peat moss, vermiculite, bark ash, starter nutrient charge (with Gypsum), slow release nitrogen and&#160;dolomitic limestone.</td>
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a technique to screen american beech for resistance to the beech scale insect (cryptococcus fagisuga lind.)

大幅に弱体化して変形している遺族の木を残しブナ樹皮病（BBD）初期の死亡率の高レベルの結果、。この疾患は、真菌のNeonectria種のいずれによる感染のエントリポイントを作成侵襲ブナカイガラムシ、 クリプトコッカスfagisugaの活動を供給することによって開始される。スケールの侵入がなければ、真菌感染症のために少し機会がある。人工的に重くBBD影響スタンドで健全な木をはびこるようにスケールの卵を使用すると、これらの木は病気の複雑な1のスケール昆虫の部分に耐性であったことを明らかにした。ここでは、フィールド内の小さな鉢植えの苗と移植片におけるスケール耐性樹木をスクリーニングするために使用することができヒューストン2による人工侵入技術をベースに我々が開発したプロトコルを提示する。スケール耐性樹木の同定はツリーをBBDの管理の重要な要素である改善プログラムや育林操作。

図6は、感受性の異なる程度を示す抵抗性苗（C）および2の影響を受けやすい苗（D、E）を示しています。人工侵入テストを設定した後の影響を受けやすく、成熟した木は57週間どのように見えるかの例を図5（b）に示します。発泡体が離れて木から剥離するときにツリーとフォームパッドの両方からデータを収集することが必須である理由で?...

Watch this Scientific Journal Video about A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind. at JoVE.com

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

ブナ樹皮病は、樹皮における真菌のエントリポイントを作成ブナスケール昆虫の活動を供給することによって開始される。カイガラムシに耐性のある木々はまた、疾患抵抗性である。ここで我々はブナの規模耐性について、個々のブナの木をスクリーニングするために開発したプロトコルを提示する。

ブナスケール昆虫への抵抗のためのアメリカのブナを（画面ための技術<em&gt;クリプトコッカスfagisuga</em&gt;リンド。）

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

a-technique-to-screen-american-beech-for-resistance-to-beech-scale

Research

JoVE Journal

Environment

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

9.5K ビュー.  US Forest Service. ブナ樹皮病は、樹皮における真菌のエントリポイントを作成ブナスケール昆虫の活動を供給することによって開始される。カイガラムシに耐性のある木々はまた、疾患抵抗性である。ここで我々はブナの規模耐性について、個々のブナの木をスクリーニングするために開発したプロトコルを提示する。 

ビデオ: A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female&#39;s seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George&#39;s Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.

A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters Homarus americanus

Fishery-induced changes to exploited crustacean fisheries, such as the American lobster fishery, could potentially influence their reproductive dynamics, leading to a reduction in mating success. This study&#39;s goal was to develop a noninvasive method for ascertaining the mating success of female lobsters that may be physiologically or functionally mature.

非侵襲的方法<em&gt;その場で</em&gt;女性アメリカンロブスターにおける交配の成功（の決定<em&gt;ロブスターのamericanus</em&gt;）

Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American ...

環境学

Video Tracking Protocol to Screen Deterrent Chemistries for Honey Bees

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey bee colony numbers have been declining in the United States and many European countries since 1947. A number of factors play a role in this decline, including the unintentional exposure of honey bees to pesticides. The development of new methods and regulations are warranted to reduce pesticide exposures to these pollinators. One approach is the use of repellent chemistries that deter honey bees from a recently pesticide-treated crop. Here, we describe a protocol to discern the deterrence of honey bees exposed to select repellent chemistries. Honey bee foragers are collected and starved overnight in an incubator 15 h prior to testing. Individual honey bees are placed into Petri dishes that have either a sugar-agarose cube (control treatment) or sugar-agarose-compound cube (repellent treatment) placed into the middle of the dish. The Petri dish serves as the arena that is placed under a camera in a light box to record the honey bee locomotor activities using video tracking software. A total of 8 control and 8 repellent treatments were analyzed for a 10 min period with each treatment was duplicated with new honey bees. Here, we demonstrate that honey bees are deterred from the sugar-agarose cubes with a compound treatment whereas honey bees are attracted to the sugar-agarose cubes without an added compound.

The loss of honey bee colonies presents a challenge to crop pollination services. Current pollinator protection practices warrant an alternative approach to minimize the contact of honey bees to harmful pesticides using repellent chemistries. Here, we provide detailed methods for a visual tracking protocol to screen deterrents for bees.

ハチミツの抑止化学物質をスクリーニングするためのビデオトラッキングプロトコル

The European honey bee, Apis mellifera L., is an economically and agriculturally important pollinator that generates billions of dollars annually. Honey ...

Lab-Scale Model to Evaluate Odor and Gas Concentrations Emitted by Deep Bedded Pack Manure

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope facilities. This protocol has been used to effectively evaluate many different bedding materials, environmental variables (temperature, humidity), and potential mitigation treatments that can improve air quality in commercial deep-bedded mono-slope facilities. The model is dynamic and allows researchers to easily collect many chemical and physical measurements from the bedded pack. Weekly measurements, collected over the course of six to seven weeks, allows sufficient time to see changes in air quality measurements over time as the bedded pack matures. The data collected from the simulated bedded packs is within the range of concentrations previously measured in commercial deep-bedded mono-slope facilities. Past studies have demonstrated that 8 - 10 experimental units per treatment are sufficient to detect statistical differences among the simulated bedded packs. The bedded packs are easy to maintain, requiring less than 10 minutes of labor per bedded packs per week to add urine, feces, and bedding. Sample collection using the gas sampling system requires 20 - 30 minutes per bedded pack, depending on the measurements that are being collected. The use of lab-scaled bedded packs allows the researcher to control variables such as temperature, humidity, and bedding source that are difficult or impossible to control in a research or commercial facility. While not a perfect simulation of &#34;real-world&#34; conditions, the simulated bedded packs serve as a good model for researchers to use to examine treatment differences among bedded packs. Several lab-scale studies can be conducted to eliminate possible treatments before trying them in a research or commercial-sized facility.

A protocol has been developed to measure gases, odors, and nutrient composition in lab-scaled bedded manure packs, which can be used to study ways to improve air quality in commercial cattle facilities using deep-bedded manure packs.

臭気と深いから放出されるガスの濃度を評価する実験室スケール モデル層状パック肥料

A lab-scaled simulated bedded pack model was developed to study air quality and nutrient composition of deep-bedded packs used in cattle mono-slope ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran insects account for a number of economically substantial pests of plants that cause damage to crops by feeding on phloem sap. The brown marmorated stink bug (BMSB), Halyomorpha halys (Heteroptera: Pentatomidae) and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae) are hemipteran insect pests introduced in North America, where they are an invasive agricultural pest of high-value specialty, row, and staple crops and citrus fruits, as well as a nuisance pest when they aggregate indoors. Insecticide resistance in many species has led to the development of alternate methods of pest management strategies. Double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) is a gene silencing mechanism for functional genomic studies that has potential applications as a tool for the management of insect pests. Exogenously synthesized dsRNA or small interfering RNA (siRNA) can trigger highly efficient gene silencing through the degradation of endogenous RNA, which is homologous to that presented. Effective and environmental use of RNAi as molecular biopesticides for biocontrol of hemipteran insects requires the in vivo delivery of dsRNAs through feeding. Here we demonstrate methods for delivery of dsRNA to insects: loading of dsRNA into green beans by immersion, and absorbing of gene-specific dsRNA with oral delivery through ingestion. We have also outlined non-transgenic plant delivery approaches using foliar sprays, root drench, trunk injections as well as clay granules, all of which may be essential for sustained release of dsRNA. Efficient delivery by orally ingested dsRNA was confirmed as an effective dosage to induce a significant decrease in expression of targeted genes, such as juvenile hormone acid O-methyltransferase (JHAMT) and vitellogenin (Vg). These innovative methods represent strategies for delivery of dsRNA to use in crop protection and overcome environmental challenges for pest management.

This article demonstrates novel techniques developed for oral delivery of double-stranded RNA (dsRNA) through the vascular tissues of plants for RNA interference (RNAi) in phloem sap feeding insects.

篩部と半翅目昆虫の植物 sap 供給の RNA 干渉を誘発する二本鎖 RNA の経口配信方法

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

海洋モデルとしてフジツボタテジマフジツボ improvisus培養と遺伝子発現

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

稚魚の行動応答プロファイルを調べるための実験プロトコル: 神経興奮剤カフェインへの応用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of emerging insects they can detect. In order to obtain precise measurement of insect emergence, it is necessary for systems to be semi-automated and able to measure large numbers of emerging insects. We addressed these issues by designing and building a system that is automated and can measure emergence of up to 1200 insects. We modified the existing &#34;falling-ball&#34; system using Arduino microcontrollers to automate data collection and expand the sample size through multiple data channels. Multiple data channels enable the user to not only increase their sample size, but also allows for multiple treatments to be run simultaneously in a single experiment. Furthermore, we created an R script to automatically visualize the data as a bubble plot, while also calculating the median day and time of emergence. The current system was designed using 3D printing so the user can modify the system to be adjusted for different species of insects. The goal of this protocol is to investigate important questions in chronobiology and stress physiology, using this precise and automated system to measure insect emergence patterns.

Measurement of insect emergence patterns requires precision. Existing systems are only semi-automated and sample size is limited. We addressed these issues by designing a system using microcontrollers to precisely measure the time of emergence of large numbers of emerging insects.

昆虫の出現パターンの検出の正確かつ自律システム

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of ...

Composition and Distribution Analysis of Bioaerosols Under Different Environmental Conditions

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, we described a protocol for multiple analyses of the biological compositions in environmental PM. Five experiments are presented: (1) PM number monitoring by using a laser particle counter; (2) PM collection by using a cyclonic aerosol sampler; (3) PM collection by using a high-volume air sampler with filters; (4) culturable microbes collection by the Andersen six-stage sampler; and (5) detection of biological composition of environmental PM by bacterial 16SrDNA and fungal ITS region sequencing. We selected hazy days and a livestock farm as two typical examples of the application in this protocol. In this study, these two sampling methods, cyclonic aerosol sampler and filter sampler, showed different sampling efficiency. The cyclonic aerosol sampler performed much better in terms of collecting bacteria, while these two methods showed the same efficiency in collecting fungi. Filter samplers can work under low temperature conditions while cyclonic aerosol samplers have a sampling limitation for temperature. A solid impacting sampler, such as an Andersen six-stage sampler, can be used to sample bioaerosols directly into the culture medium, which increases the survival rate of culturable microorganisms. However, this method mainly relies on culture while more than 99% of microbes cannot be cultured. DNA extracted from the culturable bacteria collected by the Andersen six-stage sampler and samples collected by cyclonic aerosol sampler and filter sampler were detected by bacterial 16S rDNA and fungal ITS region sequencing.All the methods above may have wide application in many fields of study, such as environmental monitoring and airborne pathogen detection. From these results, we can conclude that these methods can be used under different conditions and may help other researchers further explore the health impacts of environmental bioaerosols.

Understanding the biological composition of environmental particulate matter is important for the study of its significant impacts on human health and disease spread. Here, we used three types of bioaerosol sampling methods and a biological analysis of airborne microbes to better explore airborne microbial communities under different environmental conditions.

組成と異なる環境条件下におけるバイオエアロゾルの分布解析

Variable microorganisms in particulate matter (PM) under different environmental conditions may have significant impacts on human health. In this study, ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

噛む昆虫マンドゥカセクスタによる侵入時のジャガイモの遺伝子発現研究を簡素化する剥離葉アッセイ

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Implantation of a New Micro Acoustic Tag in Juvenile Pacific Lamprey and American Eel

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology has been identified as a way to obtain more detailed information on movement and behavior across a broader spatial scale than is possible with other known technology. The purpose of this method is to provide a detailed step by step instruction on tag implantation for both lampreys and eel. For laboratory studies using actively migrating juvenile Pacific Lamprey (120-160 mm), we determined that the presence of the tag did not alter the swimming ability between tagged and untagged Individuals or have any significant tag loss (&lt;3%). Similar results were determined during laboratory testing of Yellow phase American Eels (113-175 mm). No mortality occurred during a 38-day holding period and there was minimal tag loss (3.8%). The presence of the tag did not have any significant effect on the swimming ability or survival of tagged eels compared to untagged controls and there was minimal tag loss.

This article illustrates the procedure for implanting a micro acoustic tag in juvenile Pacific Lamprey and American Eel. Our application is based on laboratory trials, which indicate that acoustic tags can be successfully implanted with no apparent effects on swimming ability or survival and minimal tag loss.

少年太平洋ヤツメウナギやアメリカのウナギの新しいマイクロ音響タグの注入

Juvenile Pacific Lamprey and American eels were used for laboratory evaluations to determine potential effects from tag implantation. Telemetry technology ...